Volume 15, Issue 3 (vol-3 2009)                   Horizon Med Sci 2009, 15(3): 45-56 | Back to browse issues page

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Aminzadeh Gohari S, Khanahmad H, Karimipour M, Yaghmaei P, Karimi Zare S, Jamshidi F. Designing and Constructing a Plasmid with Mitochondrial Origin of Replication and Evaluating Its Replication in Human Cell Lines. Horizon Med Sci. 2009; 15 (3) :45-56
URL: http://hms.gmu.ac.ir/article-1-695-en.html
1- , hossein_khanahmad@yahoo.com
Abstract:   (12172 Views)
Background and Aim: In order to achieve a safe vector with ability to replicate autonomously in human cells by human transfactors, a recombinant plasmid with human mitochondrial origins of replication was constructed. In contrast to lentiviral and adenoviral vectors, this plasmid does not integrate into the host genome and replicates stably. Materials and Methods: Both human mitochondrial origins of replication and gfp fragments were amplified by PCR, cloned into pTZ57T/A. Hygromycin resistance gene was digested from pFBGGT. Then, four DNA fragments were subcloned into pBGGT plasmid. All steps of cloning were checked by PCR, restricted analysis and sequencing. HEK293 and ‍CHO cell lines were transfected by final plasmid (pEU). Transfected cells were checked by Fluorescence Microscope daily during 40 days. pEU and genomic DNA were extracted from transfected HEK293 treated with hygromycin. Five overlap PCRs were performed on these products to check presence of circular plasmid in transfected HEK293. Results: All steps of cloning were confirmed. Data showed that pEU did not replicate in transfected cells. Conclusion: As recombinant plasmids with both mitochondrial origins of replication did not replicate in transfected cell lines, it seems that we need to provide similar conditions of mitochondrial DNA replication in order to replicate the above vector in future experiments.
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Type of Study: Original | Subject: Internal Medicine
Received: 2010/02/16

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